The Art of Gene Expression in E. coli



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Gene expression in
E. coli




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© 2001 Webdesign by
Tilman Maier og Lars Jessen
1. Factors that influence Gene Expression
2. Expression vectors
3. Folding
4. Translocation
5. Protein stability
6. How to address the hopeless case
7. Safety
8. Links

2. Expression vectors

  • Low copy number plamids are better than high copy number plasmids (copy numbers of 5-40 is recommended). Yield of protein does not linearly correlate with copy number of a gene.

  • Examine protein production at lower copy number by using a chromosomal pcnB mutation. pcnB mutations reduce the copy number from 200 down to about 5 in pBR derived plasmids lacking the rop gene.

  • Regulated promoter - the optimum is one that is induced by a substrate such as IPTG that diffuses into the cell because then induction levels can be more easily manipulated. Inducers that are a substrate of one or more active transport systems cannot be controlled at all. These inducers will be accumulated to mM levels even when added in very low concentrations to the growth medium.

  • Co-overexpression of repressors of the promoter used can provide excellent control of transcription. Co-overexpression of activators guarantees that each promoter on the plasmids is activated.

  • Place transcription terminator at the 3' end of target gene. This avoids formation of antisense RNAs from downstream promoters operating in reverse orientation with respect to the gene that should be expressed at high levels.